The long term goal of this project has been, and remains, a detailed understanding in physical and chemical terms, of the transport mechanism of the Na/K pump. That understanding is important because the Na/K pump maintains the electrochemical gradients for Na and K ions that underlie electrical signalling in excitable cells, and coupled transport processes in most cells; the Na/K pump is also thought to mediate the therapeutic action of the cardiotonic steroid, and specific inhibitor of the pump, digoxin, still one of the most widely prescribed cardiac drugs. Charge translocation by the pump during the ion transport cycle, or accompanying partial reactions, is a fundamental feature of pump activity and not only provides a convenient and sensitive signal for determining turnover rates and rates of conformational transitions, but also sheds light on the underlying mechanisms. Because of that charge movement within the membrane's electric field, those turnover rates and the rates of partial reactions of the Na/K pump are, in general, voltage dependent. The ion transport mechanism of the Na/K pump is therefore best studied under voltage clamp conditions. The specific aims are to further characterize the charge translocating step(s) in the transport cycle by pursuing quantitative analysis of the [Na]o and [K]o dependence of pump charge movements over a range of membrane potentials, and to address the question of whether, and under what conditions, pump activity may be modulated by cellular regulatory processes. Our approach is to measure steady-state, and voltage-induced transient (pre-steady-state), pump currents in guinea- pig ventricular myocytes, used because of their high pump site density (~1200 mum-2). Myocytes are voltage clamped and internally dialyzed via wide tipped (~5 mum), low resistance (~1Momega), patch pipettes fitted with a device for rapidly exchanging the pipette solution while recording. Complementary measurements will be made using the new "giant" excised patch technique, in which steady (and, hopefully, transient) pump currents can be determined under conditions of greatly improved access to the cytoplasmic surface of the membrane, a particular advantage for investigating modulation of the Na/K pump. For modulation, we will pursue possible effects on stationary and transient pump currents of kinase-mediated phosphorylation of the pump (or closely associated regulatory molecule), or of interactions of the pump with cytoplasmic Ca ions. We will also begin structure/function studies of exogenous Na/K pumps expressed in Xenopus oocytes from injected mRNA.